pIRES2-DsRed-Express2

pIRES2-DsRed-Express2

pIRES2-DsRed-Express2载体基本信息

pIRES2-DsRed-Express2载体质粒图谱和多克隆位点信息

pIRES2-DsRed-Express2载体简介

pIRES2-DsRed-Express2 is a bicistronic expression vector that allows the simultaneous expression of your protein of interest and DsRed-Express2 from the same mRNA transcript.The vector is designed to allow efficient flow cytometric detection of transiently transfected mammalian cells expressing either DsRed-Express2 and a protein of interest, or DsRedExpress2 alone (at lower signal intensity).

DsRed-Express2 is a variant of the Discosoma sp. red fluorescent protein, DsRed (1).It retains the fast maturation and high photostability characteristic of its predecessor,DsRed-Express (2), and has been engineered (through additional amino acid substitutions) for increased solubility (3). Although it most likely forms the same tetrameric structure as wild-type DsRed, DsRed-Express2 displays a greatly reduced tendency to aggregate,resulting in reduced cyto- and phototoxicity, and making DsRed-Express2 much better suited for in vivo applications involving sensitive cells, such as primary or stem cells.DsRed-Express2 also exhibits extremely low residual green fluorescence, which allows cells expressing the protein to be effectively separated from other fluorescently labeled cell populations by flow cytometry

Bicistronic expression of the protein of interest and DsRed-Express2 is facilitated by the encephalomyocarditis virus (EMCV) internal ribosome entry site 2 (IRES2), located between the multiple cloning site (MCS) and the DsRed-Express2 coding region. This IRES allows cap-independent translation of DsRed-Express2 from an internal start site at the IRES/DsRed-Express2 junction (4, 5). Expression of the bicistronic transcript is driven by the constitutively active human cytomegalovirus immediate early promoter (PCMV IE) located just upstream of the MCS.

SV40 polyadenylation signals downstream of the DsRed-Express2 coding sequence direct proper processing of the 3' ends of the bicistronic mRNA. The vector backbone also contains an SV40 origin for replication in mammalian cells expressing the SV40 large T antigen, a pUC origin of replication for propagation in E. coli,and an f1 origin for single-stranded DNA production. This vector also has a neomycin-resistance cassette (Neor) that allows G418 selection of stably transfected eukaryotic cells (6). This cassette consists of the SV40 early promoter, a Tn5 kanamycin/neomycin resistance gene, and herpes simplex virus thymidine kinase (HSV TK) polyadenylation signals. A bacterial promoter upstream of this cassette allows kanamycin resistance in E. coli.

Use

When cloning into the pIRES2 DsRed-Express2 MCS, your gene of interest must contain an initiation codon (ATG) and a stop codon. Cells expressing the gene of interest can be quickly identified by screening for DsRed-Express2 fluorescence.pIRES2 DsRed-Express2 and its derivatives can be introduced into mammalian cells using any standard transfection method. Cells expressing DsRed-Express2 (excitation and emission maxima: 554 and 591,respectively) can be detected by flow cytometry or microscopy 24 hr after transfection. However, in some cases, up to 48 hr may be required for detection. If required, stable transfectants can be selected using G418. Please refer to the Living Colors® User Manual provided with this vector for additional information on detection of DsRed-Express2.

pIRES2-DsRed-Express2载体序列

ORIGIN

    1 TAGTTATTAA TAGTAATCAA TTACGGGGTC ATTAGTTCAT AGCCCATATA TGGAGTTCCG

   61 CGTTACATAA CTTACGGTAA ATGGCCCGCC TGGCTGACCG CCCAACGACC CCCGCCCATT

  121 GACGTCAATA ATGACGTATG TTCCCATAGT AACGCCAATA GGGACTTTCC ATTGACGTCA

  181 ATGGGTGGAG TATTTACGGT AAACTGCCCA CTTGGCAGTA CATCAAGTGT ATCATATGCC

  241 AAGTACGCCC CCTATTGACG TCAATGACGG TAAATGGCCC GCCTGGCATT ATGCCCAGTA

  301 CATGACCTTA TGGGACTTTC CTACTTGGCA GTACATCTAC GTATTAGTCA TCGCTATTAC

  361 CATGGTGATG CGGTTTTGGC AGTACATCAA TGGGCGTGGA TAGCGGTTTG ACTCACGGGG

  421 ATTTCCAAGT CTCCACCCCA TTGACGTCAA TGGGAGTTTG TTTTGGCACC AAAATCAACG

  481 GGACTTTCCA AAATGTCGTA ACAACTCCGC CCCATTGACG CAAATGGGCG GTAGGCGTGT

  541 ACGGTGGGAG GTCTATATAA GCAGAGCTGG TTTAGTGAAC CGTCAGATCC GCTAGCGCTA

  601 CCGGACTCAG ATCTCGAGCT CAAGCTTCGA ATTCTGCAGT CGACGGTACC GCGGGCCCGG

  661 GATCCGCCCC TCTCCCTCCC CCCCCCCTAA CGTTACTGGC CGAAGCCGCT TGGAATAAGG

  721 CCGGTGTGCG TTTGTCTATA TGTTATTTTC CACCATATTG CCGTCTTTTG GCAATGTGAG

  781 GGCCCGGAAA CCTGGCCCTG TCTTCTTGAC GAGCATTCCT AGGGGTCTTT CCCCTCTCGC

  841 CAAAGGAATG CAAGGTCTGT TGAATGTCGT GAAGGAAGCA GTTCCTCTGG AAGCTTCTTG

  901 AAGACAAACA ACGTCTGTAG CGACCCTTTG CAGGCAGCGG AACCCCCCAC CTGGCGACAG

  961 GTGCCTCTGC GGCCAAAAGC CACGTGTATA AGATACACCT GCAAAGGCGG CACAACCCCA

 1021 GTGCCACGTT GTGAGTTGGA TAGTTGTGGA AAGAGTCAAA TGGCTCTCCT CAAGCGTATT

 1081 CAACAAGGGG CTGAAGGATG CCCAGAAGGT ACCCCATTGT ATGGGATCTG ATCTGGGGCC

 1141 TCGGTACACA TGCTTTACAT GTGTTTAGTC GAGGTTAAAA AAACGTCTAG GCCCCCCGAA

 1201 CCACGGGGAC GTGGTTTTCC TTTGAAAAAC ACGATGATAA TATGGCCACA ACCATGGATA

 1261 GCACTGAGAA CGTCATCAAG CCCTTCATGC GCTTCAAGGT GCACATGGAG GGCTCCGTGA

 1321 ACGGCCACGA GTTCGAGATC GAGGGCGAGG GCGAGGGCAA GCCCTACGAG GGCACCCAGA

 1381 CCGCCAAGCT GCAGGTGACC AAGGGCGGCC CCCTGCCCTT CGCCTGGGAC ATCCTGTCCC

 1441 CCCAGTTCCA GTACGGCTCC AAGGTGTACG TGAAGCACCC CGCCGACATC CCCGACTACA

 1501 AGAAGCTGTC CTTCCCCGAG GGCTTCAAGT GGGAGCGCGT GATGAACTTC GAGGACGGCG

 1561 GCGTGGTGAC CGTGACCCAG GACTCCTCCC TGCAGGACGG CACCTTCATC TACCACGTGA

 1621 AGTTCATCGG CGTGAACTTC CCCTCCGACG GCCCCGTAAT GCAGAAGAAG ACTCTGGGCT

 1681 GGGAGCCCTC CACCGAGCGC CTGTACCCCC GCGACGGCGT GCTGAAGGGC GAGATCCACA

 1741 AGGCGCTGAA GCTGAAGGGC GGCGGCCACT ACCTGGTGGA GTTCAAGTCA ATCTACATGG

 1801 CCAAGAAGCC CGTGAAGCTG CCCGGCTACT ACTACGTGGA CTCCAAGCTG GACATCACCT

 1861 CCCACAACGA GGACTACACC GTGGTGGAGC AGTACGAGCG CGCCGAGGCC CGCCACCACC

 1921 TGTTCCAGTA GGCGGCCGCG ACTCTAGATC ATAATCAGCC ATACCACATT TGTAGAGGTT

 1981 TTACTTGCTT TAAAAAACCT CCCACACCTC CCCCTGAACC TGAAACATAA AATGAATGCA

 2041 ATTGTTGTTG TTAACTTGTT TATTGCAGCT TATAATGGTT ACAAATAAAG CAATAGCATC

 2101 ACAAATTTCA CAAATAAAGC ATTTTTTTCA CTGCATTCTA GTTGTGGTTT GTCCAAACTC

 2161 ATCAATGTAT CTTAAGGCGT AAATTGTAAG CGTTAATATT TTGTTAAAAT TCGCGTTAAA

 2221 TTTTTGTTAA ATCAGCTCAT TTTTTAACCA ATAGGCCGAA ATCGGCAAAA TCCCTTATAA

 2281 ATCAAAAGAA TAGACCGAGA TAGGGTTGAG TGTTGTTCCA GTTTGGAACA AGAGTCCACT

 2341 ATTAAAGAAC GTGGACTCCA ACGTCAAAGG GCGAAAAACC GTCTATCAGG GCGATGGCCC

 2401 ACTACGTGAA CCATCACCCT AATCAAGTTT TTTGGGGTCG AGGTGCCGTA AAGCACTAAA

 2461 TCGGAACCCT AAAGGGAGCC CCCGATTTAG AGCTTGACGG GGAAAGCCGG CGAACGTGGC

 2521 GAGAAAGGAA GGGAAGAAAG CGAAAGGAGC GGGCGCTAGG GCGCTGGCAA GTGTAGCGGT

 2581 CACGCTGCGC GTAACCACCA CACCCGCCGC GCTTAATGCG CCGCTACAGG GCGCGTCAGG

 2641 TGGCACTTTT CGGGGAAATG TGCGCGGAAC CCCTATTTGT TTATTTTTCT AAATACATTC

 2701 AAATATGTAT CCGCTCATGA GACAATAACC CTGATAAATG CTTCAATAAT ATTGAAAAAG

 2761 GAAGAGTCCT GAGGCGGAAA GAACCAGCTG TGGAATGTGT GTCAGTTAGG GTGTGGAAAG

 2821 TCCCCAGGCT CCCCAGCAGG CAGAAGTATG CAAAGCATGC ATCTCAATTA GTCAGCAACC

 2881 AGGTGTGGAA AGTCCCCAGG CTCCCCAGCA GGCAGAAGTA TGCAAAGCAT GCATCTCAAT

 2941 TAGTCAGCAA CCATAGTCCC GCCCCTAACT CCGCCCATCC CGCCCCTAAC TCCGCCCAGT

 3001 TCCGCCCATT CTCCGCCCCA TGGCTGACTA ATTTTTTTTA TTTATGCAGA GGCCGAGGCC

 3061 GCCTCGGCCT CTGAGCTATT CCAGAAGTAG TGAGGAGGCT TTTTTGGAGG CCTAGGCTTT

 3121 TGCAAAGATC GATCAAGAGA CAGGATGAGG ATCGTTTCGC ATGATTGAAC AAGATGGATT

 3181 GCACGCAGGT TCTCCGGCCG CTTGGGTGGA GAGGCTATTC GGCTATGACT GGGCACAACA

 3241 GACAATCGGC TGCTCTGATG CCGCCGTGTT CCGGCTGTCA GCGCAGGGGC GCCCGGTTCT

 3301 TTTTGTCAAG ACCGACCTGT CCGGTGCCCT GAATGAACTG CAAGACGAGG CAGCGCGGCT

 3361 ATCGTGGCTG GCCACGACGG GCGTTCCTTG CGCAGCTGTG CTCGACGTTG TCACTGAAGC

 3421 GGGAAGGGAC TGGCTGCTAT TGGGCGAAGT GCCGGGGCAG GATCTCCTGT CATCTCACCT

 3481 TGCTCCTGCC GAGAAAGTAT CCATCATGGC TGATGCAATG CGGCGGCTGC ATACGCTTGA

 3541 TCCGGCTACC TGCCCATTCG ACCACCAAGC GAAACATCGC ATCGAGCGAG CACGTACTCG

 3601 GATGGAAGCC GGTCTTGTCG ATCAGGATGA TCTGGACGAA GAGCATCAGG GGCTCGCGCC

 3661 AGCCGAACTG TTCGCCAGGC TCAAGGCGAG CATGCCCGAC GGCGAGGATC TCGTCGTGAC

 3721 CCATGGCGAT GCCTGCTTGC CGAATATCAT GGTGGAAAAT GGCCGCTTTT CTGGATTCAT

 3781 CGACTGTGGC CGGCTGGGTG TGGCGGACCG CTATCAGGAC ATAGCGTTGG CTACCCGTGA

 3841 TATTGCTGAA GAGCTTGGCG GCGAATGGGC TGACCGCTTC CTCGTGCTTT ACGGTATCGC

 3901 CGCTCCCGAT TCGCAGCGCA TCGCCTTCTA TCGCCTTCTT GACGAGTTCT TCTGAGCGGG

 3961 ACTCTGGGGT TCGAAATGAC CGACCAAGCG ACGCCCAACC TGCCATCACG AGATTTCGAT

 4021 TCCACCGCCG CCTTCTATGA AAGGTTGGGC TTCGGAATCG TTTTCCGGGA CGCCGGCTGG

 4081 ATGATCCTCC AGCGCGGGGA TCTCATGCTG GAGTTCTTCG CCCACCCTAG GGGGAGGCTA

 4141 ACTGAAACAC GGAAGGAGAC AATACCGGAA GGAACCCGCG CTATGACGGC AATAAAAAGA

 4201 CAGAATAAAA CGCACGGTGT TGGGTCGTTT GTTCATAAAC GCGGGGTTCG GTCCCAGGGC

 4261 TGGCACTCTG TCGATACCCC ACCGAGACCC CATTGGGGCC AATACGCCCG CGTTTCTTCC

 4321 TTTTCCCCAC CCCACCCCCC AAGTTCGGGT GAAGGCCCAG GGCTCGCAGC CAACGTCGGG

 4381 GCGGCAGGCC CTGCCATAGC CTCAGGTTAC TCATATATAC TTTAGATTGA TTTAAAACTT

 4441 CATTTTTAAT TTAAAAGGAT CTAGGTGAAG ATCCTTTTTG ATAATCTCAT GACCAAAATC

 4501 CCTTAACGTG AGTTTTCGTT CCACTGAGCG TCAGACCCCG TAGAAAAGAT CAAAGGATCT

 4561 TCTTGAGATC CTTTTTTTCT GCGCGTAATC TGCTGCTTGC AAACAAAAAA ACCACCGCTA

 4621 CCAGCGGTGG TTTGTTTGCC GGATCAAGAG CTACCAACTC TTTTTCCGAA GGTAACTGGC

 4681 TTCAGCAGAG CGCAGATACC AAATACTGTT CTTCTAGTGT AGCCGTAGTT AGGCCACCAC

 4741 TTCAAGAACT CTGTAGCACC GCCTACATAC CTCGCTCTGC TAATCCTGTT ACCAGTGGCT

 4801 GCTGCCAGTG GCGATAAGTC GTGTCTTACC GGGTTGGACT CAAGACGATA GTTACCGGAT

 4861 AAGGCGCAGC GGTCGGGCTG AACGGGGGGT TCGTGCACAC AGCCCAGCTT GGAGCGAACG

 4921 ACCTACACCG AACTGAGATA CCTACAGCGT GAGCTATGAG AAAGCGCCAC GCTTCCCGAA

 4981 GGGAGAAAGG CGGACAGGTA TCCGGTAAGC GGCAGGGTCG GAACAGGAGA GCGCACGAGG

 5041 GAGCTTCCAG GGGGAAACGC CTGGTATCTT TATAGTCCTG TCGGGTTTCG CCACCTCTGA

 5101 CTTGAGCGTC GATTTTTGTG ATGCTCGTCA GGGGGGCGGA GCCTATGGAA AAACGCCAGC

 5161 AACGCGGCCT TTTTACGGTT CCTGGCCTTT TGCTGGCCTT TTGCTCACAT GTTCTTTCCT

 5221 GCGTTATCCC CTGATTCTGT GGATAACCGT ATTACCGCCA TGCAT

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